Development of Antisense HIV-1 Plasmid DNA (M. Shahabuddin and T. Bryan). In an effort to achieve efficient and long-term inhibition of viral replication and propagation, we have engineered a recombinant plasmid DNA containing several genes of HIV-1, that can trancribe antisense RNAs. HIV-1 pNL4-3 DNA was used for the construction of the recombinant antisense plasmid DNA. Intitial studies to evaluate effect of the antisense DNA against HIV-1 were performed by co-transfecting various concentrations of the anti-sense DNA with infectious viral DNA into 293 cells, a human embryonic kidney cell line, by the calcium phospate procedure. The results indicated inhibition of RT acitivty from the infectious HIV-1 DNA due to the presence of the antisense DNA. Studies are ongoing to evaluate the inhibitory effect of the recombinant anti-sense DNA in infected cells using primary HIV-1 isolates as well.Development of an HIV-1 DNA Vaccine (T. A. Galvin). The most commonly used promoter in HIV-1 vaccine plasmid DNAs are from MMTV and CMV. DNA vaccines using these promoters to express HIV-1 genes have not resulted in successful high-level and long-term immune responses. We have constructed an HIV-1 plasmid DNA containing the gag and env genes from pNL4-3 DNA and a murine retroviral promoter/enhancer. The in vitro analysis of a variety of cells transfected with the HIV-1 vaccine DNA construct indicates: correct size RNAs are transcribed from the construct by Northern blot analysis; viral proteins by Western blot analysis; and particle formation by EM. Studies are ongoing to analyze the immune responses of the HIV DNA construct in vivo in mice and monkeys.